Information about the extracellular environment is communicated to the inside of a cell by so-called signal transduction systems. Schlessinger, J., TIBS 13: 443-7 (1988); Stock, J. B., et al., Microbio Rev.53:450-490 (1989). Several classes of signal transducers are able to "transmit" a signal without internalization of the primary signal. Such signal transmission may result from binding of a ligand to a membrane-bound receptor, inducing a conformational change in the receptor.
One such signal transducer is the toxR-ctx system in Vibrio cholerae. Ctx is the gene for cholera toxin. ToxR protein is a transmembrane protein with a DNA-binding domain at its amino terminus, a strongly hydrophobic transmembrane domain, and an environmentally regulated, dimer-forming domain at its carboxyl terminus. Miller, V. L., et al. Cell 48:271-279 (1987). According to current beliefs, the hydrophobic transmembrane region directs the carboxyl terminus to the periplasm while the amino terminus remains in the cytoplasm. A change in osmolarity or temperature in the periplasm is believed to effect the capacity of the toxR protein to form dimers with its carboxyl terminus. The carboxyl dimers in turn modulate the ability of the amino terminus of toxR to bind to repetitive sequences in the ctx promoter and, thereby, modulate the ability of RNA polymerase to initiate transcription at the promoter. Id.
To test the hypothesis that toxR protein is a membrane protein, Miller et al. fused toxR protein with alkaline phosphatase (phoA). Cell 48:271-279 (1987). The phoA enzyme naturally exists as a dimer in the periplasm and the phoA-toxR fusion protein was constitutively functional with respect to both phosphatase activity and toxR activation of ctx. This result demonstrated that toxR was able to direct alkaline phosphatase to the periplasm where it was active, and that the dimerization capacity of alkaline phosphatase could direct the toxR protein to activate ctx transcription.
In a similar fusion experiment, Mekalanos et al, fused toxR to a phoA mutant that requires Zn.sup.2+ for dimerization and activity (personal communication). This fusion protein's phosphatase activity was dependent on Zn.sup.2+, as expected, but so also was activation. This result is consistent with the signal transduction model for toxR action.
Chimeric signal transduction systems have been constructed in prokaryotic cells with different bacterial systems (Utsumi, J., et al.,Science 245:1246-1249), in tissue culture cells with different eukaryotic hormone receptors (Riedel, H., et al., EMBO J. 8:2943-2954 (1989)), and in tissue culture cells with a bacterial aspartate binding domain and the insulin receptor (Moe, G. E., .et al., Proc. Natl. Acad. Sci 86:5683-7 (1989)). To date, however, no such system has employed the toxR receptor for V. cholerae.